目的探讨融合蛋白CTLA4.FasL对肝前体细胞(LEPCs)增殖分化潜能的影响及抑制异种排斥反应的效应。方法克隆CTLA4.FasL基因,构建携带CTLA4.FasL基因与红色荧光蛋白(mCherry)双顺反子结构的重组慢病毒载体Lv-CTLA4.FasL-IRES-mCherry。优化慢病毒感染LEPCs的条件,建立高表达CTLA4.FasL的LEPCs,荧光显微镜观察mCherry的表达,Western blot检测CTLA4.FasL的表达,酶联免疫吸附测定法(ELISA)测定细胞培养上清中CTLA4.FasL的浓度,水溶性四氮唑(WST-1)法检测细胞的增殖活性,Real time PCR(RT-PCR)检测干细胞相关基因CK19和c-Kit mRNA的表达,5-溴脱氧尿嘧啶核苷(Brdu)掺入法测定CTLA4.FasL-LEPCs在异种混合淋巴细胞培养体系中对大鼠淋巴细胞增殖的抑制作用。结果构建重组慢病毒载体Lv-CTLA4.FasL-IRES-mCherry,病毒滴度为2×108 TU/mL。在感染复数(multiplicity of infection,MOI)为10,聚凝胺质量浓度是5μg/mL时,感染效率约90%。Western blot证实了CTLA4.FasL的表达,在细胞培养上清中其质量浓度约为(0.72±0.10)μg/mL。CTLA4.FasL-LEPCs细胞增殖活性未受到影响,CK19和c-Kit基因mRNA表达水平无变化。CTLA4.FasL-LEPCs细胞可显著抑制大鼠淋巴细胞的增殖(P<0.05)。结论构建成功的重组慢病毒载体Lv-CTLA4.FasL-IRES-mCherry可介导CTLA4.FasL基因在LEPCs中高效表达;CTLA4.FasL可有效地抑制异种排斥反应,同时不损害LEPCs增殖活性和分化潜能。
Objective: Hepatic progenitor cell transplantation has shed light on the treatment of liver failure. The present study was designed to evaluate whether xenogeneic liver epithelial progenitor cells(LEPCs) transplantation could promote liver recovery in a rat model of acute liver failure. The engraftment and hepatocytic differentiation of transplanted hepatic progenitor cells in the rat spleen was also investigated. Methods: LEPCs were propagated in vitro for long and transduced with lentiviral vector carrying mCherry gene. Intraperitoneal injection of CCl4 followed by 2/3 partial hepatectomy three days later were used to establish rat models of acute liver failure. Rats were intrasplenically injected with mCherry modified LEPCs(n=20, 1×107cells/0.5 mL) or the same volume of medium(n=20). Serum liver enzymes(ALT, AST) and liver histology were evaluated for 21 days after transplantation. The engraftment of transplanted LEPCs in the spleens was tested by polymerase chain reaction(PCR) amplification targeting mCherry gene. The differentiation into hepatocytic lineage of transplanted LEPCs was investigated using immunohistochemistry staining against Alb. Results: LEPCs were effectively transduced with lentiviral vector showing a transduction efficiency of 90%. Compared with control, cell-injected group displayed significantly lower levels of ALT and AST(P<0.05) and better histological features including less swelling change and hepatocyte death. PCR amplification of mCherry sequences confirmed the engraftment of LEPCs in the spleens. Alb-positive cells first appeared 5 days after cell transplantation and the number of Alb-positive cells increased substantially(P<0.05), which revealed the hepatocytic differentiation process. Conclusion: Xenogeneic hepatic progenitor cells can engraft and differentiate into hepatocytes in the splenic parenchyma. Intrasplenic delivery of hepatic progenitor cells ameliorates CCl4 /partial hepatectomy-induced liver injury in rats.
WAN ZhenLü YiZHANG XiaogangZHENG Xing-longWU WanquanMA JiaWANG Haohua
